CN114428130A - Method for obtaining lipid-lowering potential marker and metabolic pathway of walnut green seedcase polyphenol extract - Google Patents
Method for obtaining lipid-lowering potential marker and metabolic pathway of walnut green seedcase polyphenol extract Download PDFInfo
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- CN114428130A CN114428130A CN202111572448.7A CN202111572448A CN114428130A CN 114428130 A CN114428130 A CN 114428130A CN 202111572448 A CN202111572448 A CN 202111572448A CN 114428130 A CN114428130 A CN 114428130A
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- walnut green
- polyphenol extract
- green husk
- liver
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- 125000000430 tryptophan group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12 0.000 description 1
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 239000000341 volatile oil Substances 0.000 description 1
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Abstract
The invention discloses a liver potential metabolic marker for reducing lipid of a walnut green husk polyphenol extract and a method for obtaining a metabolic pathway of the liver potential metabolic marker, wherein 18 potential markers are screened from liver metabolites which are obviously influenced by the walnut green husk polyphenol extract, and a metabolic fingerprint is obtained from the liver metabolites; analyzing related metabolic pathways of the two groups of different metabolites through MetPA to obtain 12 liver metabolic pathways after intervention of the walnut green husk polyphenol extract. Detecting high-fat diet-induced hyperlipidemic rat liver metabolites by using a UPLC/Q-TOF-MS technology to obtain related liver potential markers of the walnut green husk polyphenol extract for reducing fat, analyzing content changes of the liver potential markers of the walnut green husk polyphenol extract for reducing fat by using the technology, and analyzing metabolic pathways of the potential markers to obtain the metabolic pathways of the walnut green husk polyphenol extract for reducing fat. Comprehensively and comprehensively evaluating the lipid-lowering action mechanism of the walnut green husk polyphenol extract from the overall level, and providing a basis for developing and utilizing walnut green husk.
Description
Technical Field
The invention belongs to the technical field of biological resource utilization, and particularly relates to a method for obtaining a potential lipid-lowering marker and a metabolic pathway of a walnut green husk polyphenol extract.
Background
Abnormal metabolism or transport of fat in the human body can cause the blood total cholesterol TC or triglyceride TG to be too high or the high density lipoprotein cholesterol to be too low. The damage to the body is hidden, gradual, progressive and systemic, and if the intervention is not performed in time, a series of complications can be further caused, and the human health is seriously harmed. Therefore, it is very important to find drugs that can intervene in abnormal processes of lipid metabolism and elucidate the mechanism of action. Metabolomics is the science of studying the collection of all small molecule metabolites in cells, tissues or organs. The purpose is to quantitatively analyze the content of metabolites in the organism, further help to explain the functions of genes, reveal the association among various metabolic networks and help researchers to understand the organism more systematically. The comprehensive and integral characteristics of metabonomics can explain the disease occurrence and the lipid-lowering action mechanism of bioactive substances by observing the change of endogenous micromolecule metabolites after a biological system is stimulated or interfered.
The exocarpium Juglandis Immaturus is Juglans regia (Jugladaceae) plantJuglans regiaL. the epicarp of the fruit is mostly thrown away as waste in the production and processing process, which causes resource waste and environmental pollution. The exocarpium Juglandis Immaturus mainly contains quinones, flavonoids, phenols, fatty acids, steroids, diarylheptanoids, terpenes, polysaccharides, volatile oil, etc. The walnut green seedcase recorded in the medical book has the effects of clearing away heat and toxic materials, dispelling wind, treating tinea, relieving pain, stopping dysentery and the like, and also has the effects of resisting tumors, resisting oxidation, inhibiting bacteria, relieving pain and the like. Application of modern biotechnology in researching walnut greenThe active ingredients in the skin and the action mechanism thereof are beneficial to the development and utilization of agricultural and forestry waste resources. With the development of ecological industry, the walnut cultivation area and the walnut yield are rapidly increased, the walnut green husk resources are very rich, but the resource utilization level is low, so that huge waste is caused. Therefore, as a resource with good medicinal value, how to better extract and treat the effective components in the walnut green seedcase and effectively utilize the effective components is worth deep research.
Disclosure of Invention
The first purpose of the invention is to provide a potential lipid-lowering marker of a walnut green husk polyphenol extract.
The invention also aims to provide a method for obtaining the potential lipid-lowering marker and metabolic pathway of the walnut green husk polyphenol extract.
The first object of the invention is realized by that the walnut green husk polyphenol extract is a lipid-lowering liver potential marker which is screened from the liver by being significantly influenced by the walnut green husk polyphenol extract to 18 potential markers, wherein 13 markers are selected from the liver in a positive ion mode and 5 markers are selected from the liver in a negative ion mode, wherein the potential markers comprise riboflavin, pipecolic acid, tyrosine, L-methionine, lysine, L-leucocine, hypoxanthine, glycophosphocholine, glutathione, Gamma-Glu-Leu, cytosine nucleoside, 9 h-xanthine, R-carnitine, UDP-glucose, opthalmic acid, N-Acetylglutamic acid, glucose 6-phosphate, ADP;
the marker obtains a metabolic fingerprint from liver metabolites of a rat with hyperlipidemia induced by high-fat diet and a dry prognosis walnut green husk polyphenol extract, and potential lipid-lowering markers of 18 walnut green husk polyphenol extracts are screened out and identified by multivariate statistical analysis;
analyzing related metabolic pathways of two groups of different metabolites by MetPA to obtain 12 liver metabolic pathways after intervention by walnut green husk polyphenol extracts, namely D-glutamine and D-glutamic acid metabolism, biosynthesis of phenylalanine, tyrosine and tryptophan, glutathione metabolism, taurine and hypotaurine metabolism, biosynthesis of valine, leucine and isoleucine, interconversion of pentose and gluconate, alanine, aspartic acid and glutamic acid metabolism, nicotinic acid and nicotinamide metabolism, arginine and proline metabolism, tryptophan metabolism, tyrosine metabolism and starch and sucrose metabolism.
The other purpose of the invention is realized by a method for preparing lipid-lowering potential markers and metabolic pathways by using the walnut green husk polyphenol extract, which is characterized by comprising the following specific operation steps:
(1) preparing a walnut green seedcase polyphenol extract: sun drying and crushing walnut green seedcase, sieving with a 80-mesh sieve, weighing a certain amount of walnut green case powder, adding 50-70% ethanol according to a liquid-material ratio of 20:1, performing reflux extraction for 80-100 min, controlling the temperature at 50-60 ℃, filtering, extracting for 2 times, combining filtrates, performing vacuum concentration, and performing freeze drying to obtain a walnut green case polyphenol extract;
(2) preparing high-fat feed: the high-fat feed is prepared by uniformly mixing 60-75 parts by weight of basic feed, 5-15 parts by weight of yolk powder, 5-15 parts by weight of lard, 1-4 parts by weight of cholesterol, 0.6-1.0 part by weight of pig bile salt, 5-15 parts by weight of cane sugar and 0.1-0.3 part by weight of salt, and drying at 45-55 ℃;
(3) preparation of animal model: healthy male SD rats 72 with a weight of 130-150 g were randomly divided into 2 groups after adaptive feeding for one week: one group was 60 model groups, and the other group was 12 normal control groups; the rats in the normal control group were fed with normal diet, and the rats in the model group were fed with homemade high-fat diet to construct a model of hyperlipidemic rats. Molding for 4 weeks, randomly extracting 6 rats in the model group and the control group respectively, collecting blood, detecting total cholesterol TC and triglyceride TG in the blood, and judging whether the molding of the hyperlipemia rat model is successful or not; randomly dividing the hyperlipidemia rats successfully modeled into a model group, a positive medicine group and a walnut green husk polyphenol extract dry preparation group;
(4) collecting and pretreating rat liver: at the end of the test, the rat is fasted for 12 hours without water prohibition, the rat is dissected after anesthesia by 10% chloral hydrate, the liver is quickly taken out, the rat is rinsed by 0.9% physiological saline, blood stains and dirt are removed, connective tissues, adipose tissues and the like are removed, the rat is sucked dry, the sample is divided into small blocks of about 1g by sterile scissors, the small blocks are subpackaged in sterile centrifuge tubes, the sterile centrifuge tubes are immediately placed in liquid nitrogen for quick freezing for at least 15min and then are placed at-80 ℃ for storage; taking a liver sample to obtain a sample to be detected, and detecting a liver metabolite by using an UPLC/Q-TOF-MS technology;
(5) LC-MS detection: chromatographic conditions are as follows: the instrument adopts WatersACQUITYLC, an ACQUITYUPLC BEH C181.7 mu m (2.1X 100 mm) chromatographic column is used, the temperature of an automatic sample injector is set to be 4 ℃, gradient elution is carried out by injecting 10 mu L of sample at the flow rate of 0.25mL/min and the column temperature of 40 ℃, and the mobile phase is A phase 0.1% formic acid water to B phase 0.1% formic acid acetonitrile; the gradient elution procedure is 0-1 min, 2% of phase B; 1-9.5 min, 2-50% of phase B; 9.5-14 min, 50-98% of phase B; 14-15 min, 98% of phase B; 15-15.5 min, 98-2% of phase B; 15.5-17 min, 2% of phase B; mass spectrum conditions: the instrument uses ThermoLTQerbitrapXL electrospray ion source ESI and a positive and negative ion ionization mode, wherein the positive ion spray voltage is 4.80kV, the negative ion spray voltage is 4.50kV, the sheath gas is 45arb, and the auxiliary gas is 15 arb; the method comprises the following steps of carrying out full scanning at a resolution of 60000 at a capillary temperature of 325 ℃, a capillary voltage of 35V/-15V and a tube lens voltage of 50V/-50V, wherein the scanning range is 50-1000, CID is adopted for secondary cracking, the collision voltage is 30eV, unnecessary MS/MS information is removed by dynamic exclusion (repeated counting is 2), and the dynamic exclusion time is set to be 15 s;
(6) data preprocessing: converting the obtained original data into an mzXML format (XCMS input file format) by Proteowizard software (v3.0.8789), and performing peak identification, peak filtering and peak alignment by using an XCMS program package of R (v3.3.2); the main parameters are bw =5, ppm =15, peak = c (10,120), mzwid =0.015, mzdiff =0.01, and method = "centWave". Obtaining a data matrix comprising information such as a mass-to-nuclear ratio (m/z), retention time, peak area and the like; 4535 precursor molecules are obtained in the positive ion mode, 4188 precursor molecules are obtained in the negative ion mode, and data are exported to excel for subsequent analysis; in order to enable data of different magnitudes to be compared, peak area batch normalization is carried out on the data;
(7) screening and pathway analysis of potential biomarkers: sample data were first subjected to adaptive conversion (UV) and then subjected to multivariate statistical analysis.
The rat liver metabolite of high-fat diet-induced hyperlipidemia is detected by using the UPLC/Q-TOF-MS technology to obtain the liver potential marker related to lipid reduction of the walnut green husk polyphenol extract, the content change of the liver potential marker of lipid reduction of the walnut green husk polyphenol extract is further analyzed by the technology, and the metabolic pathway analysis is carried out on the potential marker to obtain the lipid reduction metabolic pathway of the walnut green husk polyphenol extract. Comprehensively evaluating the lipid-lowering action mechanism of the walnut green husk polyphenol extract from the whole level, and providing a basis for developing and utilizing walnut green husk.
Drawings
FIG. 1 is a total base peak flow chromatogram in liver of a control group, a hyperlipidemic rat model group and a walnut green seedcase polyphenol extract intervention group based on UPLC/Q-TOF-MS technology, wherein: 1-1 is in positive ion mode; 1-2 is in negative ion mode, CD is in normal group, HFD is in model group, and WGH is in walnut green husk polyphenol extract group.
FIG. 2 is a diagram of the metabolic pathway analyzed using the metaboanalyst platform of the present invention, in which: a is D-glutamine and D-glutamic acid metabolism, B is biosynthesis of phenylalanine, tyrosine and tryptophan, C is glutathione metabolism, D is taurine and hypotaurine metabolism, E is biosynthesis of valine, leucine and isoleucine, F is interconversion of pentose and gluconate, G is alanine, aspartic acid and glutamic acid metabolism, H is niacin and nicotinamide metabolism, I is arginine and proline metabolism, J is tryptophan metabolism, K is tyrosine metabolism, and L is starch and sucrose metabolism.
Detailed Description
The present invention is further described with reference to the following drawings and examples, but the present invention is not limited thereto in any way, and any modifications or alterations made according to the technical teaching and teaching of the present invention are within the scope of the present invention.
The lipid-lowering liver potential marker of the walnut green husk polyphenol extract and the method for obtaining the lipid-lowering potential marker and metabolic pathway of the walnut green husk polyphenol extract are realized by the following specific operation steps:
the preparation method of the walnut green husk polyphenol extract comprises the steps of drying and crushing walnut green husks, sieving with a 80-mesh sieve, weighing a certain amount of walnut green husk powder, adding 50% ethanol in a liquid-material ratio of 20:1, carrying out reflux extraction for 80min, controlling the temperature at 50 ℃, extracting for 2 times, combining filtrates, carrying out vacuum concentration, and carrying out freeze drying to obtain the walnut green husk polyphenol extract.
The high-fat feed is prepared by uniformly mixing 67 parts by weight of basic feed, 10 parts by weight of egg yolk powder, 10 parts by weight of lard, 2 parts by weight of cholesterol, 0.8 part by weight of pig bile salt, 10 parts by weight of sucrose and 0.2 part by weight of table salt, and drying at 45-55 ℃ after preparation and molding.
The animal model is prepared by randomly dividing 72 healthy male SD rats with the weight of 130-150 g into 2 groups after adaptive feeding for one week: one group was 60 model groups, and the other group was 12 normal control groups; the rats in the normal control group were fed with normal diet, and the rats in the model group were fed with homemade high-fat diet to construct a model of hyperlipidemic rats. Modeling for 4 weeks, randomly drawing 6 rats in each of a model group and a control group, collecting blood, detecting total cholesterol TC and triglyceride TG in the blood, and judging whether modeling of the hyperlipemia rat model is successful or not; after the molding is successful, the model group is randomly divided into model groups to be fed with high-fat feed and perfused with physiological saline with the same quantity as the stomach; feeding high-fat feed and gastric administration simvastatin 40mg/kg.d by the positive medicine group; feeding high-fat feed and intragastrically perfusing the walnut green husk polyphenol extract into intervention groups according to different doses, wherein the high dose is 600mg/kg.d, the medium dose is 300mg/kg.d, and the low dose is 150 mg/kg.d; feeding normal control group with common feed, and performing intragastric administration with normal saline; during the period, the feed is freely eaten, the temperature of the feeding environment is 242 ℃, the day and night are alternated for 12h, and the administration is continuously carried out for 11 weeks.
Collecting and pretreating rat livers, wherein in the final stage of experiment, the rats are fasted and are not forbidden to be water for 12 hours, the rats are dissected after being anesthetized by 10% chloral hydrate, the livers are quickly taken out, the rats are rinsed by 0.9% of normal saline, blood stains and dirt are removed, connective tissues, adipose tissues and the like are removed, the rat livers are sucked dry, a sample is divided into about 1g of small blocks by sterile scissors, the small blocks are packaged in a sterile centrifuge tube, the centrifuge tube is immediately placed in liquid nitrogen for quick freezing for at least 15 minutes, and the small blocks are placed at-80 ℃ for storage; taking a liver sample of 100mg in a 2mL tube, adding 1000 mu L of methanol-water 4:1 mixed solution, and adding 5 steel balls at the temperature of minus 20 ℃; placing into a high-throughput tissue grinder at 70Hz for 1 min; grinding, treating in an ultrasonic machine at room temperature for 30min, standing on ice for 30min, centrifuging at 14000RPM and 4 ℃ for 10min, collecting supernatant of 800 μ L, transferring into a new centrifugal tube of 1.5mL, and concentrating the sample with a vacuum centrifugal concentrator; dissolving the sample with 400 μ L methanol water solution with concentration of 1:1 at 4 deg.C, filtering with 0.22 μm membrane to obtain sample to be detected, and detecting liver metabolite by UPLC/Q-TOF-MS technology.
The LC-MS detection and the chromatographic conditions are as follows: the instrument adopts WatersACQUITYLC, an ACQUITYUPLC BEH C181.7 mu m (2.1X 100 mm) chromatographic column is used, the temperature of an autosampler is set to be 4 ℃, gradient elution is carried out by feeding 10 mu L of sample at the column temperature of 40 ℃ at the flow rate of 0.25mL/min, and mobile phases are A phase 0.1% formic acid water and B phase 0.1% formic acid acetonitrile; the gradient elution procedure is 0-1 min, 2% of phase B; 1-9.5 min, 2-50% of phase B; 9.5-14 min, 50-98% of phase B; 14-15 min, 98% of phase B; 15-15.5 min, 98-2% of phase B; 15.5-17 min, 2% of phase B; mass spectrum conditions: the instrument uses ThermoLTQerbitrapXL electrospray ion source ESI and a positive and negative ion ionization mode, wherein the positive ion spray voltage is 4.80kV, the negative ion spray voltage is 4.50kV, the sheath gas is 45arb, and the auxiliary gas is 15 arb; the capillary temperature is 325 ℃, the capillary voltage is 35V/-15V, the tube lens voltage is 50V/-50V, full scanning is carried out at the resolution of 60000, the scanning range is 50-1000, secondary cracking is carried out by CID, the collision voltage is 30eV, unnecessary MS/MS information is removed by dynamic exclusion (repeated counting is 2), and the dynamic exclusion time is set to be 15 s.
The data preprocessing is to convert the obtained original data into an mzXML format (XCMS input file format) through Proteowizard software (v3.0.8789), and to perform peak identification, peak filtering and peak alignment by using an XCMS program package of R (v3.3.2); the main parameters are bw =5, ppm =15, peak = c (10,120), mzwid =0.015, mzdiff =0.01, and method = "centWave". Obtaining a data matrix comprising information such as a mass-to-nuclear ratio (m/z), retention time, peak area and the like; 4535 precursor molecules are obtained in the positive ion mode, 4188 precursor molecules are obtained in the negative ion mode, and data are exported to excel for subsequent analysis; to enable comparison of data of different magnitudes, batch normalization of peak areas was performed on the data.
Screening and pathway analysis of the potential biomarkers, firstly adopting adaptive conversion (UV) to sample data, then carrying out multivariate statistical analysis to screen differential metabolites under the conditions that p-value is less than or equal to 0.05 and fold _ change is more than or equal to 1.5 or less than or equal to 0.667 and one-way ANOVA p-value is less than or equal to 0.05, and searching for differential metabolites; identification of metabolites is firstly confirmed according to accurate molecular weight (the molecular weight error is less than 20ppm), and then the metabolites are obtained by confirming and annotating a Human Metamolome Database (HMDB), a Metlin, a massbank, a LipidMaps, an mzclound and a Smart nuclear organism (Smart Nuclear) self-built standard Database according to MS/MS fragment mode alignment; the 18 potential markers screened from the liver are obviously influenced by the walnut green husk polyphenol extract, wherein 13 potential markers are selected under a positive ion mode, and 5 potential markers are selected under a negative ion mode, and comprise riboflavin, pipecolic acid, tyrosine, L-methionine, lysine, L-leucine, hypoxanthine, glycophosphocholine, glutathione, Gamma-Glu-Leu, cytosine nucleoside, 9 h-xanthine, (R) carnitine, UDP-glucose, opthalmic acid, N-Acetylglutamic acid, glucose 6-phosphate and ADP.
Analyzing related metabolic pathways of two groups of different metabolites through a MetPA database, wherein an adopted data analysis algorithm is hyper-geometric test, and a pathway topological structure adopts Relative-beta energy center; data between groups are expressed as S + -D, and the data of each group are analyzed by mean + -standard deviation using software GraphPad Prism 7.0 statistics
SD), multiple inter-group comparisons using ONE-WAY anove; p is a radical of formula<0.05 as the statistically different criteria, p<0.01 is a very significant standard of difference; after the intervention of the walnut green husk polyphenol extract, a liver sample obtains 12 paths, namely D-glutamine and D-glutamic acid metabolism, biosynthesis of phenylalanine, tyrosine and tryptophan, glutathione metabolism, taurine and hypotaurine metabolism, biosynthesis of valine, leucine and isoleucine, mutual conversion of pentose and gluconate, alanine, aspartic acid and glutamic acid metabolism, nicotinic acid and nicotinamide metabolism, arginine and proline metabolism, tryptophan metabolism, tyrosine metabolism and starch and sucrose metabolism.
Example 1
Sun drying exocarpium Juglandis Immaturus, pulverizing, sieving with 80 mesh sieve, weighing a certain amount of exocarpium Juglandis Immaturus powder, adding 50% ethanol at a liquid-material ratio of 20:1, reflux extracting for 80min at 50 deg.C for 2 times, mixing filtrates, vacuum concentrating, and freeze drying to obtain exocarpium Juglandis Immaturus polyphenol extract; the high-fat feed is prepared by uniformly mixing 60 parts by weight of basic feed, 15 parts by weight of yolk powder, 15 parts by weight of lard, 4 parts by weight of cholesterol, 1.0 part by weight of pig bile salt, 15 parts by weight of cane sugar and 0.3 part by weight of table salt, and drying at 55 ℃ after preparation and molding; rats were fed with the high-fat diet according to the procedure for preparation of animal models to construct hyperlipidemia rat models. After molding for 4 weeks, collecting blood, detecting total cholesterol TC and triglyceride TG in the blood, and determining that molding of the rat model with hyperlipidemia is successful; the stomach-filling walnut green husk polyphenol extract comprises 600mg/kg.d of high content, 300mg/kg.d of medium content and 150mg/kg.d of low content; feeding normal control group with common feed, and performing intragastric administration with normal saline; during the period, the feed is freely eaten, the temperature of the feeding environment is 242 ℃, the day and night are alternated for 12h, and the administration is continuously carried out for 11 weeks. At the end of the test, the rat is fasted for 12 hours without water prohibition, the rat is dissected after anesthesia by 10% chloral hydrate, the liver is quickly taken out, rinsed by 0.9% normal saline, blood stain and dirt are removed, connective tissue, adipose tissue and the like are removed, sucked dry, the sample is divided into small blocks of about 1g by sterile scissors, the small blocks are subpackaged in a sterile centrifuge tube, immediately placed in liquid nitrogen and quickly frozen for at least 15 minutes, and then placed at-80 ℃ for storage; taking a liver sample 100mg in a 2mL tube, adding 1000. mu.L of methanol-water mixed solution (4:1, -20 ℃), and adding 5 steel balls; placing into a high-throughput tissue grinder at 70Hz for 1 min; grinding, treating in an ultrasonic machine at room temperature for 30min, standing on ice for 30min, centrifuging at 14000RPM and 4 ℃ for 10min, collecting supernatant of 800 μ L, transferring into a new centrifugal tube of 1.5mL, and concentrating the sample with a vacuum centrifugal concentrator; dissolving the sample with 400 μ L methanol water solution with concentration of 1:1 at 4 deg.C, filtering with 0.22 μm membrane to obtain sample to be detected, and detecting liver metabolite by UPLC/Q-TOF-MS technology. 18 potential markers which are obviously influenced by the walnut green husk polyphenol extract are obtained through LC-MS detection, data preprocessing, screening of potential biomarkers and pathway analysis, wherein 13 potential markers are obtained in a positive ion mode, and 5 potential markers are obtained in a negative ion mode and comprise riboflavin, pipecolic acid, tyrosine, L-methionine, lysine, L-leucocine, hypoxanthine, glycophosphocholine, glutathione, Gamma-Glu-Leu, cytosine nucleoside, 9 h-xanthine, (R) carnitine, UDP-glucose, opthalmic acid, N-Acetylglutamic acid, glucose 6-phosphate and ADP. Meanwhile, 12 liver sample passages after intervention of the walnut green husk polyphenol extract are obtained, namely D-glutamine and D-glutamic acid metabolism, biosynthesis of phenylalanine, tyrosine and tryptophan, glutathione metabolism, taurine and hypotaurine metabolism, biosynthesis of valine, leucine and isoleucine, mutual conversion of pentose and gluconate, alanine, aspartic acid and glutamic acid metabolism, nicotinic acid and nicotinamide metabolism, arginine and proline metabolism, tryptophan metabolism, tyrosine metabolism and starch and sucrose metabolism.
Example 2
Sun drying and pulverizing exocarpium Juglandis Immaturus, sieving with 80 mesh sieve, weighing a certain amount of exocarpium Juglandis Immaturus powder, adding 70% ethanol with volume fraction at a liquid-material ratio of 20:1, reflux extracting for 100min at 60 deg.C for 2 times, mixing filtrates, vacuum concentrating, and freeze drying to obtain exocarpium Juglandis Immaturus polyphenol extract; the high-fat feed is prepared by uniformly mixing 75 parts by weight of basic feed, 5 parts by weight of yolk powder, 5 parts by weight of lard, 1 part by weight of cholesterol, 0.6 part by weight of pig bile salt, 5 parts by weight of cane sugar and 0.1 part by weight of table salt, and drying at 45 ℃ after preparation and molding; rats were fed with the high fat diet in the same manner as in example 1 to construct a hyperlipidemic rat model. After molding for 4 weeks, collecting blood, detecting total cholesterol TC and triglyceride TG in the blood, and determining that molding of the rat model with hyperlipidemia is successful; the stomach-filling walnut green husk polyphenol extract comprises 600mg/kg.d of high content, 300mg/kg.d of medium content and 150mg/kg.d of low content; feeding normal control group with common feed, and perfusing with normal saline with equal amount; during the period, the feed is freely eaten, the temperature of the feeding environment is 242 ℃, the day and night are alternated for 12h, and the administration is continuously carried out for 11 weeks. At the end of the test, the rat is fasted for 12 hours without water prohibition, the rat is dissected after anesthesia by 10% chloral hydrate, the liver is quickly taken out, the rat is rinsed by 0.9% physiological saline, blood stains and dirt are removed, connective tissues, adipose tissues and the like are removed, the rat is sucked dry, the sample is divided into small blocks of about 1g by sterile scissors, the small blocks are subpackaged in sterile centrifuge tubes, the sterile centrifuge tubes are immediately placed in liquid nitrogen for quick freezing for at least 15min and then are placed at-80 ℃ for storage; taking a liver sample to obtain a sample to be detected, and detecting the liver metabolite by using the UPLC/Q-TOF-MS technology. The 18 potential markers which are obviously influenced by the walnut green seedcase polyphenol extract are obtained through LC-MS detection, data preprocessing, screening of potential biomarkers and pathway analysis. And obtaining 12 liver sample passages intervened by the walnut green seedcase polyphenol extract.
Example 3
Sun drying and pulverizing exocarpium Juglandis Immaturus, sieving with 80 mesh sieve, weighing a certain amount of exocarpium Juglandis Immaturus powder, adding 60% ethanol with volume fraction at a liquid-material ratio of 20:1, reflux extracting for 90min at 55 deg.C for 2 times, mixing filtrates, vacuum concentrating, and freeze drying to obtain exocarpium Juglandis Immaturus polyphenol extract; the high-fat feed is prepared by uniformly mixing 72 parts by weight of basic feed, 10 parts by weight of yolk powder, 10 parts by weight of lard, 2 parts by weight of cholesterol, 0.8 part by weight of pig bile salt, 10 parts by weight of cane sugar and 0.21 part by weight of table salt, and drying at 50 ℃ after preparation and forming; rats were fed with the high-fat diet in the same manner as in example 1 to construct a hyperlipidemic rat model. After molding for 4 weeks, collecting blood, detecting total cholesterol TC and triglyceride TG in the blood, and determining that molding of the rat model with hyperlipidemia is successful; the stomach-filling walnut green husk polyphenol extract comprises 600mg/kg.d of high content, 300mg/kg.d of medium content and 150mg/kg.d of low content; feeding normal control group with common feed, and perfusing with normal saline with equal amount; during the period, the feed is freely eaten, the temperature of the feeding environment is 242 ℃, the day and night are alternated for 12h, and the administration is continuously carried out for 11 weeks. At the end of the test, the rat is fasted for 12 hours without water prohibition, the rat is dissected after anesthesia by 10% chloral hydrate, the liver is quickly taken out, the rat is rinsed by 0.9% physiological saline, blood stains and dirt are removed, connective tissues, adipose tissues and the like are removed, the rat is sucked dry, the sample is divided into small blocks of about 1g by sterile scissors, the small blocks are subpackaged in sterile centrifuge tubes, the sterile centrifuge tubes are immediately placed in liquid nitrogen for quick freezing for at least 15min and then are placed at-80 ℃ for storage; taking a liver sample to obtain a sample to be detected, and detecting the liver metabolite by using the UPLC/Q-TOF-MS technology. The 18 potential markers which are obviously influenced by the walnut green seedcase polyphenol extract are obtained through LC-MS detection, data preprocessing, screening of potential biomarkers and pathway analysis. And obtaining 12 liver sample passages intervened by the walnut green seedcase polyphenol extract.
Table 1 is a detailed OPLS-DA model verification parameter table of liver samples based on UPLC/Q-TOF-MS technology. In the table, CD is a normal group, HFD is a model group, and WGH is a walnut green seedcase polyphenol extract group. Wherein: re, the number of principal components; R2X, model (for X-variable data set) interpretability; R2Y, model (for Y-variable data sets) interpretability; q2, model predictability.
TABLE 1 liver sample detailed OPLS-DA model verification parameter table based on UPLC/Q-TOF-MS technology
Table 2 is a table of potential biomarkers for liver samples based on the UPLC/Q-TOF-MS technique of the present invention. In the table, mz: a mass to nuclear ratio; rt: retention time, unit s; identity: the result of the identification; form: ionization mode, [ M + H ] + is positive ion mode, [ M-H ] -is negative ion mode; an exact Mass: precise molecular weight; formula: a theoretical molecular formula; KEGG is KEGG compound number.
TABLE 2 liver sample potential biomarker Table based on UPLC/Q-TOF-MS technique
Claims (8)
1. The lipid-lowering liver potential metabolic marker of the walnut green husk polyphenol extract is characterized in that the marker is screened from the liver metabolites to 18 potential markers which are significantly influenced by the walnut green husk polyphenol extract, wherein 13 markers are selected in a positive ion mode, and 5 markers are selected in a negative ion mode and comprise riboflavin, pipecolic acid, tyrosine, L-methionine, lysine, L-leucosine, hypoxanthine, glycophosphocholine, glutathione, Gamma-Glu-Leu, cytosine nucleoside, 9 h-xanthine, R-carnitine, UDP-glucose, opthalmic acid, N-Acetylglutamic acid, glucose 6-phosphate and ADP;
the marker obtains a metabolic fingerprint from liver metabolites of a rat with hyperlipidemia induced by high-fat diet and a dry prognosis walnut green husk polyphenol extract, and potential lipid-lowering markers of 18 walnut green husk polyphenol extracts are screened out and identified by multivariate statistical analysis;
analyzing related metabolic pathways of two groups of different metabolites by MetPA to obtain 12 liver metabolic pathways after intervention by walnut green husk polyphenol extracts, namely D-glutamine and D-glutamic acid metabolism, biosynthesis of phenylalanine, tyrosine and tryptophan, glutathione metabolism, taurine and hypotaurine metabolism, biosynthesis of valine, leucine and isoleucine, interconversion of pentose and gluconate, alanine, aspartic acid and glutamic acid metabolism, nicotinic acid and nicotinamide metabolism, arginine and proline metabolism, tryptophan metabolism, tyrosine metabolism and starch and sucrose metabolism.
2. A method for obtaining lipid-lowering potential markers and metabolic pathways of the walnut green husk polyphenol extract of claim 1, which is characterized by comprising the following specific operation steps:
(1) preparation of walnut green husk polyphenol extract
Sun drying and crushing walnut green seedcase, sieving with a 80-mesh sieve, weighing a certain amount of walnut green case powder, adding 50-70% ethanol according to a liquid-material ratio of 20:1, performing reflux extraction for 80-100 min, controlling the temperature at 50-60 ℃, extracting for 2 times, combining filtrates, performing vacuum concentration, and performing freeze drying to obtain a walnut green case polyphenol extract;
(2) preparation of high fat feed
The high-fat feed is prepared by uniformly mixing 60-75 parts by weight of basic feed, 5-15 parts by weight of yolk powder, 5-15 parts by weight of lard, 1-4 parts by weight of cholesterol, 0.6-1.0 part by weight of pig bile salt, 5-15 parts by weight of cane sugar and 0.1-0.3 part by weight of salt, and drying at 45-55 ℃;
(3) preparation of animal models
Healthy male SD rats 72 with a weight of 130-150 g were randomly divided into 2 groups after adaptive feeding for one week: one group was 60 model groups, and the other group was 12 normal control groups; feeding the normal control group rats with a common feed, and feeding the model group rats with a self-made high-fat feed to construct a hyperlipidemia rat model; randomly drawing 6 rats of a model group and a control group respectively after molding for 4 weeks, collecting blood, detecting total cholesterol TC and triglyceride TG in the blood, judging whether molding of the rat model with the hyperlipidaemia is successful or not, and randomly dividing the rat model with the hyperlipidaemia, which is successfully molded, into a model group, a positive medicine group and a walnut green husk polyphenol extract dry preparation group;
(4) collection and pretreatment of rat liver
At the end of the test, the rat is fasted for 12 hours without water prohibition, the rat is dissected after anesthesia by 10% chloral hydrate, the liver is quickly taken out, the rat is rinsed by 0.9% physiological saline, blood stains and dirt are removed, connective tissues, adipose tissues and the like are removed, the rat is sucked dry, the sample is divided into small blocks of about 1g by sterile scissors, the small blocks are subpackaged in sterile centrifuge tubes, the sterile centrifuge tubes are immediately placed in liquid nitrogen for quick freezing for at least 15min and then are placed at-80 ℃ for storage; taking a liver sample to obtain a sample to be detected, and detecting a liver metabolite by using an UPLC/Q-TOF-MS technology;
(5) LC-MS detection
Chromatographic conditions are as follows: the instrument adopts WatersACQUITYLC, an ACQUITYUPLC BEH C181.7 mu m (2.1X 100 mm) chromatographic column is used, the temperature of an autosampler is 4 ℃, the sample injection is carried out by 10 mu L at the flow rate of 0.25mL/min and the column temperature of 40 ℃, and the mobile phase is A phase 0.1 percent formic acid water and B phase 0.1 percent formic acid acetonitrile; the gradient elution procedure is 0-1 min, 2% of phase B; 1-9.5 min, 2-50% of phase B; 9.5-14 min, 50-98% of phase B; 14-15 min, 98% of phase B; 15-15.5 min, 98-2% of phase B; 15.5-17 min, 2% of phase B; mass spectrum conditions: the instrument uses ThermoLTQerbitrapXL electrospray ion source ESI and a positive and negative ion ionization mode, wherein the positive ion spray voltage is 4.80kV, the negative ion spray voltage is 4.50kV, the sheath gas is 45arb, and the auxiliary gas is 15 arb; the method comprises the following steps of carrying out full scanning at a resolution of 60000 at a capillary temperature of 325 ℃, a capillary voltage of 35V/-15V and a tube lens voltage of 50V/-50V, wherein the scanning range is 50-1000, CID is adopted for secondary cracking, the collision voltage is 30eV, unnecessary MS/MS information is removed by dynamic exclusion (repeated counting is 2), and the dynamic exclusion time is set to be 15 s;
(6) data pre-processing
Converting the obtained original data into an mzXML format through Proteowizard software, and performing peak identification, peak filtration and peak alignment by using an XCMS program package of R; the main parameters are bw =5, ppm =15, peak = c (10, 120), mzwid =0.015, mzdiff =0.01, and method = "centWave", so as to obtain a data matrix including information of a nucleus ratio m/z, retention time, peak area and the like; 4535 precursor molecules are obtained in the positive ion mode, 4188 precursor molecules are obtained in the negative ion mode, and data are exported to excel for subsequent analysis; in order to enable data of different magnitudes to be compared, peak area batch normalization is carried out on the data;
(7) screening and pathway analysis of potential biomarkers
Sample data were first subjected to adaptive conversion (UV) and then subjected to multivariate statistical analysis.
3. The method for obtaining lipid-lowering potential markers and metabolic pathways of the walnut green husk polyphenol extract as claimed in claim 2, wherein the preparation of the walnut green husk polyphenol extract in the step (1) is that the walnut green husk is dried in the sun, crushed and sieved by a 80-mesh sieve, a certain amount of walnut green husk powder is weighed according to a liquid-material ratio of 20:1, added with 50% ethanol by volume fraction and extracted under reflux for 80min, the temperature is controlled at 50 ℃, filtered, extracted for 2 times, the filtrate is combined, concentrated under vacuum, and freeze-dried to obtain the walnut green husk polyphenol extract.
4. The method for obtaining lipid-lowering potential markers and metabolic pathways of the walnut green husk polyphenol extract as claimed in claim 2, wherein the high-fat feed in the step (2) is prepared by uniformly mixing 67 parts by weight of basic feed, 10 parts by weight of egg yolk powder, 10 parts by weight of lard oil, 2 parts by weight of cholesterol, 0.8 part by weight of pig bile salt, 10 parts by weight of sucrose and 0.2 part by weight of salt, preparing and molding, and drying at 45-55 ℃.
5. The method for obtaining lipid-lowering potential markers and metabolic pathways of the walnut green husk polyphenol extract as claimed in claim 2, wherein the model group is randomly divided into a model group after the model building in the step (3) is successful, and fed with high-fat feed and perfused with normal saline with the same amount as that of the stomach; feeding high-fat feed and gastric administration simvastatin 40mg/kg.d by the positive medicine group; the walnut green husk polyphenol extract is fed to a group for intervention with high-fat feed and is perfused with the walnut green husk polyphenol extract in different doses, wherein the high dose is 600mg/kg.d, the medium dose is 300mg/kg.d, and the low dose is 150 mg/kg.d; feeding normal control group with common feed, and perfusing with normal saline with equal amount; during the period, the food is freely eaten, the feeding environment temperature is 242 ℃, the day and night are alternated for 12h, and the administration is continuously carried out for 11 weeks.
6. The method for obtaining lipid-lowering potential markers and metabolic pathways of the walnut green husk polyphenol extract as claimed in claim 2, wherein the step (4) comprises taking 100mg of the liver sample in a 2mL tube, adding 1000. mu.L of a 4:1 mixed solution of methanol and water, and adding 5 steel balls at-20 ℃; placing into a high-throughput tissue grinder at 70Hz for 1 min; grinding, treating in an ultrasonic machine at room temperature for 30min, standing on ice for 30min, centrifuging at 14000RPM and 4 ℃ for 10min, collecting supernatant of 800 μ L, transferring into a new centrifugal tube of 1.5mL, and concentrating the sample with a vacuum centrifugal concentrator; dissolving the sample with 400 μ L methanol water solution with concentration of 1:1 at 4 deg.C, filtering with 0.22 μm membrane to obtain sample to be detected, and detecting liver metabolite by UPLC/Q-TOF-MS technology.
7. The method for obtaining potential markers and metabolic pathways for lipid lowering of the walnut green husk polyphenol extract as claimed in claim 2, wherein in the step (7), differential metabolite screening is performed under the conditions that p-value is less than or equal to 0.05+ fold _ change is greater than or equal to 1.5 or less than or equal to 0.667 and one-way ANOVA p-value is less than or equal to 0.05 to search for differential metabolites; identification of metabolites is firstly confirmed according to accurate molecular weight (the molecular weight error is less than 20ppm), and then metabolites are obtained by comparing Human Metamolome Database (HMDB), Metlin, massbank, LipidMaps, mzclound and intelligent nuclear organism Smart Nucle self-constructed standard Database confirmation annotation according to MS/MS fragment mode; the screening of the liver to 18 potential markers is obviously influenced by the walnut green husk polyphenol extract, wherein 13 potential markers are selected in a positive ion mode, and 5 potential markers are selected in a negative ion mode, and comprise riboflavin, pipecolic acid, tyrosine, L-methionine, lysine, L-leucine, hypoxanthine, glycophosphocholine, glutathione, Gamma-Glu-Leu, cytosine nucleoside, 9 h-xanthine, (R) carnitine, UDP-glucose, opthalmic acid, N-Acetylglutamic acid, glucose 6-phosphate and ADP.
8. The method for detecting potential lipid-lowering markers and metabolic pathways of walnut green husk polyphenol extract as claimed in claim 2, wherein the step (7) is to analyze the relevant metabolic pathways of two groups of different metabolites by a MetPA database, the adopted data analysis algorithm is hyper-geometric test, and the path topology adopts Relative-beta metabolism; the data among the groups are expressed as S +/-D, the software GraphPad Prism 7.0 is used for statistical analysis, the data among the groups are expressed as mean +/-standard deviation (x +/-S), and ONE-WAY ANOVET is adopted for multiple comparison among the groups; p is less than 0.05, which is the standard of statistical difference, and p is less than 0.01, which is the standard of significant difference; after the intervention of the walnut green husk polyphenol extract, a liver sample obtains 12 paths, namely D-glutamine and D-glutamic acid metabolism, biosynthesis of phenylalanine, tyrosine and tryptophan, glutathione metabolism, taurine and hypotaurine metabolism, biosynthesis of valine, leucine and isoleucine, mutual conversion of pentose and gluconate, alanine, aspartic acid and glutamic acid metabolism, nicotinic acid and nicotinamide metabolism, arginine and proline metabolism, tryptophan metabolism, tyrosine metabolism and starch and sucrose metabolism.
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